The hepatitis B virus (HBV) is a noncytopathic, hepatotropic DNA virus that causes acute and chronic hepatitis and kills a million people each year from cirrhosis of the liver and hepatocellular carcinoma. Currently approved therapies for the treatment of chronic hepatitis B include two classes of drugs: interferon and a small number of nucleos(t)ide analogues. However, the indication and response to interferon therapy is poor and associated with severe adverse effects and nucleos(t)ide analogues therapy is frequently associated with the emergence of single- and cross-drug-resistant HBV mutants. Thus, developing a new interferon- and/or nucleos(t)ide analogue-independent therapy is the long-term objective of this application. Antisense-mediated degradation of viral RNA represents such a novel class of antiviral therapy and this approach has been successfully pursued by our collaborator Isis Pharmaceuticals and resulted in a commercialized drug (fomivirsen) targeting cytomegalovirus. Using the same principal, our collaborators at Isis Pharmaceuticals have now identified lead candidate antisense molecules that efficiently inhibit HBV replication in cell culture. Studying the HBV life cycle and its inhibition by antiviral drugs has been and still is hampered by the lack of suitable and widely available in vivo HBV replication and infection systems. I have extensive experience using HBV transgenic mice that express and replicate HBV from an integrated viral genome to study viral gene expression, replication, virus production and inhibition of these steps by interferon-mediated antiviral mechanism. Furthermore, together with our collaborators at the Salk Institute, we have recently demonstrated efficient HBV and hepatitis C virus infection in a novel chimeric mouse model repopulated with human hepatocytes that allows us to study all steps of the viral life cycle during long-term infections. The goal of this application is to use these in vivo HBV replication and infection model systems to further evaluate and improve the therapeutic value and characteristics of the lead candidate antisense molecules targeting HBV. In specific aim 1 we will establish the lead candidate's toxicity and pharmacokinetic profile, demonstrate in vivo activity, and identify the most potent candidates. Specific aim 2 is designed to determine the long-term tolerability and efficacy of the lead candidates in suppressing HBV gene expression and replication in the HBV transgenic mice. In specific aim 3 the antiviral efficacy of the leads and the potential for emergence of drug resistance will be tested in the context of a bonefide HBV infection initiated with a monoclonal viral isolate using human hepatocyte repopulated chimeric mice. Finally, in specific aim 4 we will determine the leads antiviral efficacy against infection with patient sera containing isolates of different HBV genotypes and sera from patients that experienced virological breakthrough on antiviral therapy. Together, these studies will be instrumental in introducing antisense based therapy as a novel approach to combat chronic HBV infection.